Liquid chromatography (LC) is an extremely important analytical technique which is used for the separation, identification, and quantitation of an analyte of interest even if present in a complex mixture of different sample constituents. During LC the chemical components in a mixture were carried through a stationary phase by the flow of a liquid mobile phase. Separation in liquid chromatography is achieved by means of differences in the interactions of the analytes with both the mobile and stationary phases. As the skilled artisan appreciates, both a stationary phase and a mobile phase appropriate to the analytes under investigation have to be chosen. In addition, the user will identify chromatographic conditions appropriate to maintain the sharpness of analyte bands as a sample moves through the stationary phase column to the detector.
High performance liquid chromatography, also known as high pressure liquid chromatography, abbreviated as HPLC, is a special form of liquid chromatography and nowadays used frequently in biochemistry and analytical chemistry. The analyte is forced through a column of the stationary phase in a liquid (mobile phase) at high pressure which decreases the time the separated components remain on the stationary phase, and thus the time they have to diffuse within the column. This leads to narrower peaks in the resulting chromatogram and thence to better resolution and sensitivity as compared to LC.
The mobile phase is chosen to ensure solubility of the sample solutes. For the stationary phase, preferably microparticulate silica (bare or chemically modified) is used because its high surface area accentuates the differences in solute-stationary phase interactions. The use of a stationary phase that interacts strongly with solutes relative to solute mobile-phase interactions will result in very long retention times, a situation which is not analytically useful. Hence the stationary phase must be selected so as to provide weak to moderate solute interactions relative to those in the mobile phase. As a consequence, the nature of the solute governs the type of LC selected. The stronger interactions should occur in the mobile phase to ensure sample solubility and ready elution, while the stationary phase should be responsive to more subtle differences among the solutes. For example, polar neutral compounds are usually better analyzed using a polar mobile phase together with a nonpolar stationary phase that distinguishes subtle differences in the dispersive character of the solutes. One of the powerful aspects of HPLC is that the mobile phase can be varied to alter the retention mechanism. Modifiers can be added to the mobile phase to control retention. For example, pH is an important variable in aqueous mobile phases.
Five general classes of LC can be distinguished:
1. Normal-phase chromatography calls for the use of a polar stationary phase in conjunction with a non-polar (dispersive) mobile phase.
2. Reversed-phase chromatography, the opposite possibility, calls for the use of a non-polar stationary phase and a polar mobile phase (composed of one or more of the polar solvents, e.g., water, methanol, acetonitrile, and tetrahydrofuran).
3. Ion-exchange chromatography involves ionic interactions. In this case the mobile phase must support ionization to ensure solubility of ionic solutes. The stationary phase must also be partially ionic to promote some retention. Consequently, the interactions with the stationary phase are strong, and this is usually reflected in longer analysis times and broad peaks.
4. Size-exclusion chromatography involves separations based on molecular size alone and ideally requires that there be no energetic interaction of the solutes with the stationary phase.
5. Affinity chromatography is based on a specific interaction, e.g., between the members of a specific binding pair like antigen and corresponding antibody or receptor and corresponding ligand. For example, a first partner of a binding pair is bound to an appropriate stationary phase and used to capture the second partner of the binding pair. The second partner can be released and isolated by appropriate means.
The general classification of separation principles given above is not, exhaustive and therefore is non-limiting. There are other separation principles which can be used for the separation of liquid samples, e.g., hydrophobic interaction chromatography, hydrophilic interaction chromatography, ion-pair chromatography, and molecular imprinted materials based separation.
The analyte of interest can be detected by any appropriate means. Appropriate and preferred detectors sense the presence of a compound passing through and provide an electronic signal to a recorder or computer data station. The output is usually in the form of a chromatogram, and a substance of interest is usually found in a certain peak. The peak area or peak height can be used to quantify the amount of analyte present in the sample investigated.
The detector for an HPLC system is the component that emits a response due to the eluting sample compound and subsequently signals a peak on the chromatogram. It is positioned immediately posterior to the stationary phase in order to detect the compounds as they elute from the column. The bandwidth and height of the peaks may usually be adjusted using the coarse and fine tuning controls, and the detection and sensitivity parameters may also be controlled by the skilled artisan. There are many types of detectors that can be used with HPLC. Some of the more common detectors include: refractive index (RI), ultra-violet (UV), fluorescent, radiochemical, electrochemical, near-infra red (near-IR), mass spectrometry (MS), nuclear magnetic resonance (NMR), and light scattering (LS).
Refractive index (RI) detectors measure the ability of sample molecules to bend or refract light. This property for each molecule or compound is called its refractive index. For most RI detectors, light proceeds through a bi-modular flow-cell to a photodetector. One channel of the flow-cell directs the mobile phase passing through the column while the other directs, only the mobile phase. Detection occurs when the light is bent due to samples eluting from the column, and this is read as a disparity between the two channels.
Fluorescent detectors measure the ability of a compound to absorb then re-emit light at given wavelengths. Each compound has a characteristic fluorescence. The excitation source passes through the flow-cell to a photodetector while a monochromator measures the emission wavelengths.
Radiochemical detection involves the use of radiolabeled material, usually tritium (3H) or carbon-14 (14C). It operates by detection of fluorescence associated with beta-particle ionization, and it is most popular in metabolite research.
Electrochemical detectors measure compounds that undergo oxidation or reduction reactions. This is usually accomplished by measuring gain or loss of electrons from migrating samples as they pass between electrodes at a given difference in electrical potential.
Mass spectrometry is an analytical technique used to measure the mass-to-charge ratio (m/z or m/q) of ions. It is most generally used to analyze the composition of a physical sample by generating a mass spectrum representing the masses of sample components. The technique has several applications including identifying unknown compounds by the mass of the compound and/or fragments thereof determining the isotopic composition of one or more elements in a compound, determining the structure of compounds by observing the fragmentation of the compound, quantitating the amount of a compound in a sample using carefully designed methods (mass spectrometry is not inherently quantitative), studying the fundamentals of gas phase ion chemistry (the chemistry of ions and neutrals in vacuum), and determining other physical, chemical or even biological properties of compounds with a variety of other approaches.
A mass spectrometer is a device used for mass spectrometry, and it produces a mass spectrum of a sample to analyze its composition. This is normally achieved by ionizing the sample and separating ions of differing masses and recording their relative abundance by measuring intensities of ion flux. A typical mass spectrometer comprises three parts: an ion source, a mass analyzer, and a detector.
The kind of ion source is a contributing factor that strongly influences-what types of samples can be analyzed by mass spectrometry. Electron ionization and chemical ionization are used for gases and vapors. In chemical ionization sources, the analyte is ionized by chemical ion-molecule reactions during collisions in the source. Two techniques often used with liquid and solid biological samples include electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI). Other techniques include fast atom bombardment (FAB), thermospray, atmospheric pressure chemical ionization (APCI), secondary ion mass spectrometry (SIMS), and thermal ionisation.
Liquid-chromatography-tandem-mass spectrometry (LC-MS/MS) has been introduced in clinical chemistry (Vogeser M., Clin. Chem. Lab. Med. 41 (2003) 117-126). Advantages of this technology are high analytical specificity and accuracy and the flexibility in the development of reliable analytical methods. In contrast to gas chromatography mass spectrometry (GC-MS) as the traditional mass spectrometric technology in clinical chemistry. LC-MS/MS has been shown to be a robust technology, allowing its application also in a large scale routine laboratory setting. Requirements for the preparation (clean-up) of sample material are limited compared to GC-MS; however, de-proteinizing is mandatory for small molecule target analyses.
Mere protein precipitation as presented by the state of the art may be sufficient for some LC-MS/MS methods, but in order to avoid ion-suppression effects for very sensitive methods, more efficient extraction methods are usually required (Annesley, T. M., Clin. Chem. 49 (2003) 1041-1044). “Off-line” or “on-line” solid phase extraction or solvent extraction are the techniques currently used to solve this problem. Respective time consuming manual sample preparation protocols so far represent an important limitation for the large scale routine application of LC-MS/MS in the clinical laboratory. Therefore, automation of sample preparation for LC-MS/MS is a goal that is addressed by application of different technical principles:
Samples can be loaded into 96-well plates to be submitted to batch protein precipitation by centrifugation (Vogeser, M. and Spöhrer. U. Clin. Chem. Lab. Med. 44 (2006) 1126-1130). This, however, is a discontinuous process since-plates have to be transferred into a centrifuge manually. Alternatively, filtration of precipitated samples using filtration plates and application of vacuum may be performed (Williams, M. G., et al., Biomed. Chromatogr. 17 (2003) 215-218). Mere protein precipitation, however, does not allow analyte concentration.
Solid phase extraction with extraction plates or single extraction cartridges allows full automation with a continuous work-flow from loading of samples until MS-analysis (Yang, L, et al., J. Chromatogr. B 809 (2004) 75-80; Alnouti, Y. et al., J. Chromatogr. A 1080 (2005) 99-106: Koal. T., et al., Clin. Chem. Lab. Med. 44 (2006) 299-305; Taming, J., et al., J. Pharm. Biomed. Anal. 41 (2006) 213-218.). In these methods, too, vacuum or positive air pressure has to be applied during extraction which is technically demanding.
The use of magnetic particles has been most successfully introduced to the automation of heterogenous immunoassays years ago (Porstmann, T. and Kiessig, S. T., J. Immunol. Methods 150 (1992) 5-21); respective particles used as the solid phase for extraction are ideally suited for automation, since this “solid phase” can be manipulated as a liquid. Today, this principle represents the predominant technology applied in a number automated immunoassay systems. Automated methods for DNA purification based on functionalized magnetic particles have been introduced to routine laboratories as well (Namvar, L., et al., J. Clin. Microbiol. 43 (2005) 2058-2064).
WO 2005/015216 and WO 2006/075185 disclose processes for the preparation of coated polymer particles containing superparamagnetic crystals. Porous, surface-functionalized particles are reacted with at least one polyisocyanate and at least one diol or at least one epoxide. WO 2005/015216 discloses that such beads are of utility in adsorption/desorption processes analogously to the mechanisms in (a) reversed phase chromatography or hydrophobic chromatography, and (b) hydrophobic interaction chromatography. Particularly, adsorption of a mixture of proteins to functionalized beads is described, followed by fractionation of the proteins by applying desorption buffers containing (a) increasing concentrations of acetonitrile and (b) decreasing concentrations of ammonium sulfate.
Functionalized magnetic beads for reversed phase magnetic isolation, desalting, concentration, and fractionation of complex peptide mixtures are commercially available under the trademark DYNABEADS (Dynal, Inc.) RFC18 from Invitrogen Corporation. According to the product description, magnetic separation allows fractionation of complex samples and the fractions can be applied to matrix assisted laser desorption ionization (MALDI) targets for MS analysis, or analyzed in other downstream applications such as electrospray-MS and HPLC.
Extraction protocols based on the use of magnetic particles have successfully been adapted for MALDI-TOF analyses. Zhang, X., et al., J. Biomol. Tech. 15 (2004) 167-175 disclose the processing of human plasma samples using a magnetic bead-based hydrophobic interaction chromatography resin. Villanueva, J., et al., Anal. Chem. 76 (2004) 1560-1570 disclose an automated sample preparation from human serum samples wherein peptides are captured and concentrated using a reversed-phase batch processing and magnetic particles which are surface-derivatized with reversed-phase ligands. However, the document also discloses that in some cases, prior to reversed-phase extraction, sera were additionally subjected to incubation with additives such as urea, DTT, or n-octylglucoside. Also, proteins were removed from sera by way of precipitation or filtration, or serum albumin was removed by affinity chromatography.
When considering quantitative testing, MALDI-TOF analysis has certain disadvantages. The sample material or a subtraction thereof has to be mixed with a solution of a matrix compound, and the mixture is applied to the target. After that the solvent is evaporated. Importantly, the evaporation process usually docs not lead to an even distribution of the analyte in the mixture on the target. Thus, depending on the particular spot hit by the laser beam in order to mobilize matrix and analyte material into the gas phase, the amount of analyte may differ from spot to spot. As a consequence, peak size does not reliably reflect the concentration of the analyte in the sample material or the subtraction thereof.
Aiming at quantitative detection by means of mass spectrometry, alternative ways to vaporize sample and/or analyte material are provided by atmospheric pressure ionization, e.g., electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI). However, prior to the analysis by mass spectrometry, the analyte to be quantitatively detected needs to be enriched or partially purified, particularly when the analyte is to be detected in complex sample material such as blood, serum, or plasma. To this end, the chromatographic separation step of LC-MS/MS is insufficient when performed alone. However, magnetic separation using beads with a functionalized hydrophobic surface prior to MS analysis is insufficient, too. This is especially true when quantitative analysis of analytes is intended.
When considering LC-MS/MS, in order to sufficiently reduce sample complexity and remove unwanted contaminants, the sample can be treated with a precipitant prior to the LC step to precipitate and subsequently remove, e.g., high molecular weight compounds such as proteins or carbohydrates. Also, a liquid/liquid extraction step is possible. Furthermore, the LC step can be designed as a 2-dimensional (2D) chromatography. In this case, a first chromatography using a first stationary phase is performed, and one particular fraction is subjected to a second chromatography, usually with a second stationary phase, before the analyte is injected into the MS/MS system. The theoretical solutions discussed here have disadvantages in that they are inconvenient, laborious, pose problems for automation and/or require sophisticated and expensive equipment.
The problem to be solved by the present invention was to improve sample preparation for quantitative target analysis of small molecule analytes with mass spectrometry means, that is, LC-MS/MS, whereby the analytes are extracted out of complex biological matrices like serum, plasma, whole blood, or lysed whole blood.
The analysis of peptides prepared from serum or plasma using hydrophobic magnetic particles as described in the state of the art basically targets molecules with a molecular weight greater than 700 Da (Daltons) and up to about 20,000 Da. In whole blood, plasma, and serum there is a plethora of compounds with a molecular weight below about 700 Da. Among these, peptides are just one group out of many others, including amino acids, lipids, and many different metabolites of the biochemical pathways. Thus, there exists a significant complexity of blood, plasma, and serum samples with regards to small (i.e., smaller than about 700 Da) molecular compounds.
The inventors unexpectedly found that the use of functionalized magnetic particles with a hydrophobic surface greatly enhances the process for extraction of small molecule analytes out of complex biological matrices such as whole blood, plasma, and serum. The magnetic particles according to the invention can reversibly bind low molecular weight compounds when the particles are added to the biological samples. A particular surprising finding was that the binding process is not disturbed when lipids, peptides, and proteins are abundantly present in the sample matrix. Another very surprising observation was that even very low amounts of functionalized magnetic beads are sufficient to extract and enrich low molecular weight compounds from complex sample matrices in a concentration-dependent manner. Thus, magnetic beads are much more convenient for reversed phase separation as opposed to stationary phases in the form of chromatography columns, which are much more prone to clogging when complex sample materials are processed.
The findings by the inventors proved to be especially advantageous when functionalized magnetic beads were used for the extraction of analytes from plasma, serum, and whole blood in which lipids, peptides, and proteins, among others, are particularly abundant. After the binding step and by separating the panicles with magnetic force from the remaining sample material, unbound components of the biological sample can be removed efficiently from the bound analytes. Elution of the analytes from the particles provides the desired low molecular weight compounds in a form which is sufficiently pure for analysis by mass spectrometry such as LC-MS/MS analysis. Even more surprising, functionalized magnetic particles with a hydrophobic surface can be used to extract small molecule analytes out of whole blood samples which, prior to extraction, were-subjected to hemolysis. Not only is the extraction (=sample preparation) procedure according to the invention suitable for qualitative detection of a desired analyte with a low molecular weight, surprisingly, the use of functionalized magnetic particles with a hydrophobic surface also allows quantitative detection of the analyte using mass spectrometry as a detection means.